Photoremovable caging groups find extensive use in many fields ranging from cell biology to materials science. The general requirement of UV or blue light is a significant limitation due to associated toxicity and poor tissue penetration. By contrast, light between 650 and 900 nm, often referred to as the near-IR window, is cytocompatible and has significant tissue penetration. Generally useful single photon reactions in the near-IR range would allow uncaging approaches to be applied in complex biological settings. My lab is approaching this difficult challenge by defining and then using the chemical reactions involved in the photodecomposition or photobleaching of near-IR fluorophores. These oft-encountered light-initiated processes can occur with rapid kinetics, often undesirably so. Our current efforts in this area are split in three aims. Aim 1: Development and mechanistic studies of near-IR photorelease chemistry. We have developed an uncaging reaction sequence initiated by near-IR light using readily synthesized C4'-dialkylamine-substituted heptamethine cyanines. We have shown that a variety of phenol- and amine- containing small molecules are quickly uncaged upon irradiation with low energy light. Detailed mechanistic studies involving mass spectrometry, NMR, and absorbance techniques have shown that release occurs through regioselective C-C cleavage and then hydrolysis of the C4'-amine. While the photooxidative cleavage reaction had been previously described in cyanine photobleaching literature, these efforts are the first to use it for productive application. We are currently broadening the scope of the release process and examining aspects of the mechanism in detail using computational (collaboration with Dr. Joseph Ivanic) and experimental techniques. Aim 2: Near-IR light control of gene expression. Several studies over the past 15 years have used UV light-mediated uncaging of small molecules in combination with inducible gene expression systems. Near-IR uncaging will allow these techniques to advance into increasingly complex biological settings and organismal contexts. We have shown that our approach can be used to regulate gene expression through uncaging of an estrogen-receptor antagonist in a ligand-dependent CreERT/LoxP-reporter cell line (collaboration with Dr. Susan Mackem). We are currently applying this method in increasingly complex, physiological context. These results will set the stage for further applications that combine near-IR uncaging with estrogen receptor driven protein chimeras. Aim 3: Application of uncaging reactions for targeted drug delivery. Light is used in a variety of cancer treatments ranging from established therapeutic techniques, such as PDT, to emerging areas such as fluorescence-guided surgery. We are applying our light-cleavable chemistry for targeted drug delivery (collaboration with Dr. Hisataka Kobayashi). This approach merges the unique potency of small molecule drugs with the high spatial control afforded by light release and molecular targeting. The use of tissue penetrant, cytocompatible near-IR light is critical because existing uncaging chemistries using UV or blue light would not be suitable for this application. In this area, we reported the first example of near-IR light cleavable antibody drug conjugate strategy. Antibody drug conjugates (ADCs) are new class of targeted cancer therapies that selectively deliver chemotherapeutic drugs or toxins to cancerous tissue. Existing ADCs rely on intracellular enzyme or thiol catalyzed cleavage to deliver active drug or toxin. As a consequence, uptake by benign tissue can lead to off-target drug delivery and dose limiting toxicity. Our strategy combines of existing of the existing approaches, with improved control of the critical release control. These constructs are currently being explored in preclinical models of head and neck and bladder cancer